As part of our master's thesis, we studied the mechanism of RNA interference between the fungus Verticillium nonalfalfae and hop (Humulus lupulus). Our goal was to demonstrate the silencing of a host plant gene in hop, induced by the pathogenic fungus through its milRNA via a mechanism known as "cross-kingdom" RNAi. Using the RT-qPCR method, we aimed to determine the relative expression level of the target transcript tr_001131F in the stems of non-infected and V. nonalfalfae-infected hop plants, to assess whether its expression was lower in infected plants. By testing different primer pairs, we sought to confirm whether the selected target transcript was silenced. In the resistant cultivar Wye Target, we observed statistically significant differences in the expression of the target transcript tr_001131F between infected and non-infected plants for all three primer pairs, with transcript silencing present in infected plants. Subsequently, we used the 5' RLM-RACE method to verify the cleavage site of the target transcript following the action of fungal milRNA. However, we did not confirm target cleavage using this method. The results of this study will contribute to a better understanding of the interaction between hop and the pathogenic fungus, as well as their exchange of small RNAs through the "cross-kingdom" RNAi mechanism. Additionally, the findings serve as a stepping stone for future studies on developing resistant plants and strategies to protect crops from various pathogens.
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